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Luigi is Mario's younger twin brother. Luigi usually joins and accompanies Mario on his adventures, serving as the secondary protagonist (although Mario has been known to go on adventures by himself). Through the years, Mario and Luigi have maintained a brotherly love for each other as shown in games like Luigi's Mansion and the Mario & Luigi series. In addition, the two of them are normally paired together in sports titles and spin-offs and although Luigi has shown to be slightly jealous of Mario at times (as he admits in Paper Mario), he loves his brother very much and looks up to him as a positive role model. For his part, Mario seems to allow his sibling rivalry to get the best of him when competing directly with Luigi. When Luigi receives a trophy in Mario Power Tennis, Mario is shown slapping Luigi's back and stepping on Luigi's foot intentionally. In the Mario Sports Mix press conference, Mario has stated that "Luigi loves being [his] sidekick", despite Luigi's complaining that he is "tired of being Player 2" and that it is his "time to shine".

Princess Peach is shown to be Mario's close companion since childhood. Mario saves Peach quite often and she usually will bake him a cake or kiss him upon her rescue. Since Peach's debut appearance, the two have been largely speculated to be in a romantic relationship. This is confirmed in the official European Mario website, in which Peach is listed as Mario's girlfriend.

In Luigi's Mansion, she sends Toad to help Luigi search for his brother, and in Mario Party 5, Peach and Mario are called "Cutest Couple". They also share chemistry together in Mario Superstar Baseball and Mario Super Sluggers, as they are "buddy players". Mario Power Tennis even goes as far as showing Mario himself telling Peach of his love for her in Peach's victory scene. Peach responded with a smile and blew a kiss. Also, in Mario's victory scene in Mario Power Tennis, Peach gives him a small kiss on his cheek. They both show deep devotion to each other as depicted in many of the games, especially in the Paper Mario series. In the comic "Warios Weihnachtsmärchen", published in the German Club Nintendo magazine, it is even revealed that they spent their graduation ball in school together, and that Peach decided in favor of Mario and against Wario. Mario and Peach are the Secret Staff Ghosts for Rainbow Road in Mario Kart: Double Dash!!. The official guide describe them at this: Aw, isn’t that cute? Mario and Peach are together again, zipping down the Rainbow Road and leaving the player coughing up their dust.

In the first Paper Mario, one of the Toads tells Mario to take Peach on a date to Shooting Star Summit. For the official guide of Yoshi's Island DS, in the description for the level Baby Mario and Baby Peach: Dynamic Duo, Nintendo mentioned that before the "romantic entanglements" in their adult lives, they teamed up as babies to stop evil. Also the official guide for Mario Party 8 stated that Peach is apple of Mario's eye. Rosalina in Super Mario Galaxy even referred to Peach as Mario's 'Special One'.